The Structure of FADD and Its Mode of Interaction with Procaspase-8

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1 Molecular Cell 22, , June 9, 2006 ª2006 Elsevier Inc. DOI /j.molcel The Structure of FADD and Its Mode of Interaction with Procaspase-8 Paul E. Carrington, 1,2 Cristinel Sandu, 1,2 Yufeng Wei, 1 Justine M. Hill, 1,3 Gaku Morisawa, 1 Ted Huang, 1 Evridipis Gavathiotis, 1 Yu Wei, 1 and Milton H. Werner 1, * 1 Laboratory of Molecular Biophysics The Rockefeller University 1230 York Avenue, Box 42 New York, New York Summary The structure of FADD has been solved in solution, revealing that the death effector domain (DED) and death domain (DD) are aligned with one another in an orthogonal, tail-to-tail fashion. Mutagenesis of FADD and functional reconstitution with its binding partners define the interaction with the intracellular domain of CD95 and the prodomain of procaspase-8 and reveal a self-association surface necessary to form a productive complex with an activated death receptor. The identification of a procaspase-specific binding surface on the FADD DED suggests a preferential interaction with one, but not both, of the DEDs of procaspase-8 in a perpendicular arrangement. FADD selfassociation is mediated by a hydrophobic patch in the vicinity of F25 in the DED. The structure of FADD and its functional characterization, therefore, illustrate the architecture of key components in the deathinducing signaling complex. Introduction The extrinsic cell death pathway is mediated by type I transmembrane receptors of the tumor necrosis factor (TNF) receptor superfamily (Ashkenazi and Dixit, 1998; Wu, 2004). The subgroup of TNF receptors that contain a death domain (DD) within the intracellular region have been termed death receptors (DRs). DRs engage trimeric ligands, which organize the receptors into trimers and stimulate the assembly of an intracellular complex containing the Fas-associated DD protein, FADD (Mort-1) (Boldin et al., 1995; Chinnaiyan et al., 1995), and the initiator procaspases 8 (Flice, MACH-1) (Boldin et al., 1996; Muzio et al., 1996) and 10 (Wang et al., 2001; Kischkel et al., 2001). The assembly of activated receptor, FADD, and the procaspases is commonly known as a death-inducing signaling complex or DISC. FADD is the nucleus of this assembly, as it is responsible for sensing the death stimulus at a receptor and recruiting the procaspases into the nascent DISC. FADD is constructed from two w90 amino acid motifs that are structurally similar to one another (Eberstadt et al., 1998; Jeong et al., 1999; Berglund et al., 2000) but can be readily distinguished by amino acid *Correspondence: mwerner@portugal.rockefeller.edu 2 These authors contributed equally to this work. 3 Present address: Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD 4072, Australia. sequence. The N-terminal death effector domain (DED) and the C-terminal DD each adopt a six a-helical bundle structure that is characteristic of a structural family of death motifs, which includes the caspase-recruitment domain (CARD) and the pyrin domain (Fesik, 2000). The FADD DD is responsible for receptor engagement, while the DED contains the binding site for procaspase-8 and/ or -10 (Chinnaiyan et al., 1995, 1996). However, mutagenesis of FADD (Thomas et al., 2002, 2004; Hill et al., 2004) and functional analysis in multiple contexts (Thomas et al., 2002, 2004; Sandu et al., 2006) demonstrated that the DED must be linked to the DD for a productive interaction with a DR (Thomas et al., 2002, 2004). Although FADD DD can act as an inhibitor at CD95 and DR5 under overexpression conditions in cell culture, an isolated FADD DD cannot be simultaneously shown to bind membrane bound receptor by coimmunoprecipitation (CoIP) with activated receptor (Zhang and Winoto, 1996; Sandu et al., 2006). These observations argue for a physical and functional relationship between the two domains of FADD. The three-dimensional structure of intact FADD illustrates that the DED and DD adopt a well-defined orientation in solution. Combined with functional identification of the CD95 and procaspase-8 binding surfaces under physiological conditions using FADD-deficient cells, the structure illustrates how CD95 and procaspase-8 are likely to be oriented as a building block for DISC assembly. Biochemical analysis in vitro and in cell culture reveals that the DR dependence on the FADD DED for DISC assembly is an obligatory self-association between FADD molecules. Our data define the surface of self-association to be localized to a hydrophobic patch in the vicinity of F25 in the DED, which contrasts a recent report (Muppidi et al., 2006). FADD self-associates at a surface that is independent of the binding site for procaspase-8 in the DED and is likely to occur only in the context of the activated DR at the cell membrane surface. In combination with the recently described structure of the molluscum contagiosum viral FLICE-inhibitory protein (v-flip) MC159 (Li et al., 2005; Yang et al., 2005), we describe a molecular model for the interaction between FADD and procaspase-8, which argues for a physical interaction between the FADD DED and one, but not both, of the DEDs resident in the prodomain of caspase-8. Results Structure Determination The three-dimensional structure of FADD has been determined for residues (of 208), using a single mutation, F25Y, to suppress self-aggregation of the protein at neutral ph in solution (Figure 1). The structure was solved using 2934 experimental restraints that included 1906 nuclear Overhauser effects (NOEs), J NHa coupling constants (Kuboniwa et al., 1994), D NH residual dipolar couplings (RDCs; Bax et al., 2001) collected in bacteriophage Pf1 (Hansen et al., 1998), and D NH couplings in CTAB-doped DLPC/CHAPSO bicelles (Wang et al., 1998). F and J angle restraints were

2 Molecular Cell 600 Figure 1. Three-Dimensional Structure of FADD (A) Stereo superposition of the backbone atoms for the 25 members of the FADD structure family refined in explicit solvent (Supplemental Data). (B) A ribbon representation of FADD illustrating the orthogonal, tail-to-tail orientation of the two domains in the same orientation as in (A). The twelve a helices have been numbered sequentially from the N terminus to the C terminus of the protein. (C) Side view of FADD, illustrating the shape of the molecule. (D) Sequencealignment of the FADD DED and DD. a helices are shaded gray and numbered above the sequence. Residues that permit structural superposition of the two domains are shaded green. Black dots below the sequenceindicate residuesof FADD DD implicated in the interaction with CD95 (Jeong et al., 1999; Bang et al., 2000; Hill et al., 2004). Open circles above the DED indicate residues forming the procaspase-8 binding surface (see text). Asterisks above the DED identify residues whose mutation interferes with the interaction between FADD and CD95 or DR5 (Thomas et al., 2002, 2004).

3 FADD Interaction with Procaspase included from the analysis of 13 C a/b chemical shifts using the program TALOS (Delaglio et al., 1995). To determine the structure, the unambiguously assigned NOEs and 3 J NHa coupling constants were used to fold the molecule from a fully extended chain using a protocol provided with Xplor-NIH (version ) (Schwieters et al., 2003) (see the Supplemental Data available with this article online). Following initial folding, iterative rounds of NOE assignment/refinement were conducted, and pseudopotentials were included for F, J, and c angles (Kuszewski and Clore, 2000), 3 J NHa coupling constants (Garrett et al., 1994), and 13 C a/b chemical shifts (Kuszewski et al., 1995). The radius of gyration was approximated from the number of amino acids to aid in the internal packing of each FADD domain (Kuszewski et al., 1999). The domain orientation resulting from the twomedia RDC refinement was established by alignment tensor projection analysis as described (Al-Hashimi et al., 2000). Of 270 calculated structures, the 100 lowest-energy structures having the best agreement with experimental restraints were subsequently refined in explicit solvent (Spronk et al., 2002) to improve the local geometry, electrostatics, and packing quality for each domain of the intact protein. The 25 lowest-energy structures comprise a family with a root mean square deviation (rmsd) from the mean structure of 1.0 Å for the nonhydrogen backbone atoms between residues (1.4 Å rmsd from the mean for all nonhydrogen atoms) (Table 1 and Supplemental Data). The Structure of FADD FADD is a tandem repeat of structurally homologous domains (Figures 1A and 1B) oriented with the N and C termini facing one another (Figures 1B and 1C). Although the sequence similarity between each domain is quite low (%12%), structurally conserved positions in each domain can be aligned by sequence (Figure 1D), permitting the superposition of the two domains to a rmsd of 2.6 Å over 23 C a positions. The structure of each FADD domain observed in the intact molecule is very similar to the structures observed for the isolated domains (Eberstadt et al., 1998; Jeong et al., 1999; Berglund et al., 2000). The rmsd between the FADD DED in the intact protein and the isolated DED (Eberstadt et al., 1998) is 1.79 Å. The rmsd between the FADD DD in the intact protein and the isolated FADD DD (Berglund et al., 2000) is 2.6 Å (superimposed for residues in helices 2 5), which is similar to the 2.3 Å rmsd observed between the two reported structures of the isolated FADD DD (Jeong et al., 1999; Berglund et al., 2000). The major distinction in the DD structure from the intact protein relative to the isolated domain is the connection between the FADD DED and first helix of the DD. This connection alters the position of this helix in the intact protein relative to that observed in the structures of the isolated domain. Table 1. Structural Statistics for FADD In Vacuum In Solvent b Rmsd from experimental restraints a NOEs (1906) Å Å Intraresidue (487) Å Å Sequential (588) Å Å Medium (1 <ji jj % 5) (472) Å Å Long (1 <ji jj > 5) (191) Å Å Hydrogen bond (168) Å Å F, J, c 1 (480) º º 3 J NHa (120) Hz 13 C a ppm 13 C b ppm 1 D phage NH (123) Hz Hz 1 D bicelle NH (137) Hz Hz Deviations from Idealized Covalent Geometry Bonds (Å) Angles (º) Impropers (º) Coordinate Precision c Backbone DED + DD (residues 5 183) Å Å DED (residues 5 86) Å Å DD (residues ) Å Å All nonhydrogen DED + DD (residues 5 183) Å Å DED (residues 5 86) Å Å DD (residues ) Å Å Quality Factors d Percent residues in most 79.7% 88.5% favorable Ramachandran Bad contacts/structure a Numbers in parentheses indicate total number of restraints. Deviations calculated for residues No NOEs were included for atoms separated by three chemical bonds in the refinement, with the exception of those involving backbone atoms. b Refined in explicit solvent as described in Spronk et al. (2002), using NOE, dihedral, and RDC restraints only. c Calculated with the programs PROCHECK and PROCHECK_NMR (Laskowski et al., 1996) relative to the mean structure of the 25 final family members. d Calculated with PROCHECK_NMR. Less than 1% of residues appear in generously allowed regions, and less than 0.1% of residues appear in unfavored regions of the Ramachandran plot. Bad contacts calculated with PROCHECK. The Domains of FADD Are Oriented Spin relaxation analysis of FADD (Figure S1) indicated that FADD tumbles with an apparent rotational correlation time (t c ) of ns calculated from modelfree analysis of the 15 N{ 1 H} backbone dynamics (Lipari and Szabo, 1982a, 1982b). This is comparable to the t c computed for each of the domains in the intact molecule (DED, t c is ns; DD, ns), suggesting that the individual domains of FADD tumble as a single entity in solution. A plot of R 2 /R 1 versus occurrence for each of the two FADD domains indicates that the two domains of FADD are likely to be oriented in solution (see Figure S2). A similar conclusion could be drawn from a plot of the RDCs measured in Pf1 phage as a function of occurrence (see Figure S2) (Braddock et al., 2001). Since the RDCs can report directly on the relative orientation of protein segments in solution (Al-Hashimi et al., 2000; Bax et al., 2001), a projection analysis was used to interpret the experimental data collected from two alignment media in order to determine whether a single relative orientation of the FADD domains existed for the proteins in solution (Al-Hashimi et al., 2000). To define that orientation, the structure of FADD was refined against both sets of experimental RDCs and the alignment tensor measured in bicelles projected into the

4 Molecular Cell 602 Figure 2. The Interdomain Interface of FADD (A) 13 C-edited (top) and 15 N-edited (bottom) NOESY spectra of FADD. The indicated strips were taken at the 13 C chemical shift of H59-CD2 and the 15 N chemical shift for the side chain of N155. These strips display NOEs (indicated) that could be inferred as interdomain contacts. Note that the 13 C-edited NOESY displays 13 C shifts in lieu of 1 H shifts to take advantage of the larger chemical shift range of 13 C. (B) The interface between the FADD DED (cyan) and DD (blue) is shown by a cylinder representation for one member of the structure family. The orientation of selected side chains for the entire family is shown for those residues (red) forming salt bridges and/or hydrogen bonds across the interface. Donor and acceptor atoms are shown in black. (C) Stereo representation of the interface colored as in (B) for each domain. A close-up view of the domain interface is shown as a molecular surface in stereo with selected side chains from one member of the family in red and the putative donor or acceptor atom in black. frame of reference of the phage (see Figure S2). This resulted in a single orientational solution for the domains of FADD (see Figure S2) (Al-Hashimi et al., 2000). This approach rigorously defined the relative orientation of the two FADD domains but could not define how closely packed the two domains were in the intact protein. Chemical shift degeneracies precluded the identification of many unambiguous interdomain NOEs that could help define the spatial relationship between the FADD domains. However, two unambiguous NOEs were observed (Figure 2A), as were several NOEs between the ends of the six-residue linker (residues 87 92) and each folded domain. Collectively, these restraints sterically hindered the orientation of the interdomain linker, defining the spatial relationship between the two FADD domains (Figures 2B and 2C). The small interface Figure 3. Mapping Functionally Deficient Mutants onto the Structure of FADD (A) The structure of FADD is shown in a surface representation and colored as described in Figure 1. Highlighted in red are residues whose mutation results in a loss of association with CD95. (B) Additional mutants, which reside on the opposite face of the DD (D106, K125, R142, and R146), are defective in CD95 association (Hill et al., 2004), but not TRADD association (Sandu et al., 2005), as discussed in the text. (C) Mapping of self-association-deficient mutants onto the structure of FADD. The structure of FADD has been rotated 90º about the vertical axis relative to (A). Highlighted in orange are residues of the DED whose mutation disrupts receptor interaction (Thomas et al., 2002, 2004) (see Supplemental Data). Highlighted in red are residues whose mutation disrupts the interaction with the receptor (DD mutants). Highlighted in tan are positions in the DED whose mutation disrupts both receptor and procaspase interaction (see text). (D) Further rotation of 150º about the vertical axis reveals the location of the procaspasespecific mutation sites in the FADD DED. These residues are highlighted in green and labeled (see text).

5 FADD Interaction with Procaspase Figure 4. Identification of the FADD Binding Surface for Procaspase-8 (A) GST-CD95 intracellular domain (IC) pull-down experiment with 35 S-labeled FADD and procaspase-8 (Hill et al., 2004). S12, R38, D44, and E51 were defective for the interaction with procaspase-8, but not CD95 IC. Note that a truncated form of FADD (residues 1 183) did not disrupt the interaction between FADD and CD95 or FADD and procaspase-8 in this context. Procaspase-8 failed to associate with GST-CD95 IC on its own or with GST itself (data not shown) in this assay. (B) FADD-deficient Jurkat I2.1 cells stably transfected with wild-type or mutant FADD, as indicated (each with an HA epitope tag) were assessed by CoIP following stimulation with antibody CH-11 (150 ng/ml for 3 hr). F25R was unable to associate with CD95, but D44R retains CD95 interaction despite its lower level of expression. (C) Ectopic expression of FADD D44R is robust in MCF7-Fas cells (Jaattela et al., 1995) and coimmunoprecipitates with CD95 as well as wild-type FADD in this context. (D) Flow cytometry analysis of stably transfected Jurkat I2.1 cells following CH-11 stimulation as in (B). Cells expressing FADD F25R and D44R are completely defective in programmed cell death. (E and F) To ensure that the apoptotic defect for D44R in Jurkat I2.1 cells was not an artifact of the level of protein expression, FADD or FADD D44R was ectopically expressed in the same cells, confirming that FADD D44R is defective in cell death. In this instance, the level of FADD or FADD D44R expression was robust (F). between the domains (buried accessible surface of 515 Å 2 ) is established through interactions that may be inferred between residues in the interhelical loops of a4/a5 and a10/a11 (Figures 2B and 2C). The side chains of E56 and H59 in the DED are in proximity to E154 and N155, respectively, and display NOEs between them in each case (Figure 2A). These residues probably form salt bridges or hydrogen bonds to establish the spatial relationship between the domains observed for FADD in solution. Interaction with the CD95 DD FADD has been extensively mutated in the DD in an effort to identify the surface of FADD responsible for interaction with activated CD95 (Jeong et al., 1999; Bang et al., 2000; Hill et al., 2004). The majority of FADD residues whose mutation disrupted the interaction with CD95 reside along a contiguous surface of the FADD DD (Jeong et al., 1999; Bang et al., 2000; Hill et al., 2004) (Figure 3). This surface (Figure 3A) is composed of a patch of positive charge (K110, R113, R114, R117, R127) and a predominantly nonpolar surface in the vicinity of the a11/a12 interhelical loop. An additional CD95 binding surface has been described (Hill et al., 2004) that forms a small basic patch (K125, R142, and R146) on the opposite side of the domain. Although the mutant R142E disrupts the interaction with CD95 (Hill et al., 2004), it has no effect on interaction between FADD and TRADD (Sandu et al., 2005). Thus, this patch of basic residues appears to play a role particular to receptor association, possibly forming contacts to a neighboring CD95 DD in the activated receptor complex.

6 Molecular Cell 604 Figure 5. FADD Self-Associates through Its DED (A) Flag-tagged full-length FADD was ectopically expressed in HEK293 cells with HA-tagged full-length FADD or HA-tagged individual FADD domains. HA-tagged full-length FADD and HA-tagged FADD DED coimmunoprecipitated with Flag-tagged full-length FADD, indicating that FADD can self-associate through the DED. (B) The DED surface responsible for FADD self-association was identified by mutagenesis at the indicated positions. Flag-tagged full-length FADD was used to coimmunoprecipitate mutant, HA-tagged full-length FADD, illustrating that a region that includes F25 and K33 forms the self-association surface. (C) The NMR peak patterns of the F25Y and F25Y/K33E are similar to one another, indicating only local chemical shift perturbations in the vicinity of K33E. By contrast, F25Y/R71E results in significant line broadening for many peaks in the DED and many spectral changes. The extent of shift perturbation is shown as a function of sequence in the histogram plots to the right of each spectrum. Asterisks indicate residues that lose more than 50% of the signal intensity in the double mutant relative to F25Y. The shift differences are represented as an increased thickness on a backbone worm representation of the FADD DED. Red indicates the position of the most shifted residue, yellow the secondmost shifted signals,

7 FADD Interaction with Procaspase Identification of a Procaspase-Specific Binding Surface on the DED To define the binding surface for procaspase-8, an initial screen introduced mutants into the DED that were neutral to the interaction between CD95 and FADD in a GST pull-down assay, but disrupted the interaction between CD95/FADD complexes and procaspase-8 (Figure 4A). This screen identified S12, R38, D44, and E51 as potential participants in the interaction with procaspase-8, which localized to a DED surface encompassing helices a1 and a4 (Figures 3D and 4A). F25R and K33E, two residues that reside on either end of the loop between a2 and a3, also failed to bind CD95 in this assay (data not shown). F25 and K33 were previously implicated in binding either procaspase-8 (Eberstadt et al., 1998) or the FLICE-inhibitory protein c-flip (Kaufmann et al., 2002) in a pull-down assay. To elucidate the role of these DED positions in procaspase interaction, F25R and D44R were stably transfected into FADD-deficient Jurkat I2.1 cells. CoIP with anti-cd95 demonstrated that D44R, but not F25R, retained the ability to coassociate with activated CD95 (Figure 4B), even though both mutants disrupted the ability of FADD to respond to FasL (Figure 4D). These observations suggested that F25R and D44R impact different steps in the signal transduction process. A potential issue was the relatively low level of stable D44R expression in Jurkat I2.1 cells, which could not be improved despite repeated attempts. To ensure that the loss of FasL response was not solely due to the expression level, FADD D44R was ectopically expressed in both MCF7-Fas cells (Jaattela et al., 1995) (Figure 4C) and Jurkat I2.1 cells (Figure 4F). Just as for the stably transfected cells, D44R retained the ability to coimmunoprecipitate with CD95 from MCF7-Fas cells (Figure 4C) and failed to respond to a FasL stimulus in Jurkat I2.1 cells (Figure 4E). Thus, the loss of function for D44R was most likely due to a disruption in the interaction with the procaspases, as suggested by Figures 4A and 4B. F25R, on the other hand, introduced a more fundamental defect. FADD Self-Associates through the DED F25, in conjunction with L28 and K33, forms a small cluster of DED residues between a2 and a3 whose mutation disrupts both the interaction with CD95 (Figure 4B and Sandu et al. [2006]) and the interaction with procaspase-8 or c-flip (Eberstadt et al., 1998; Kaufmann et al., 2002; Sandu et al., 2006). Mutation of Q34 and K35 also disrupts the interaction with procaspase-8 and c-flip (Kaufmann et al., 2002). We reasoned that since mutation of F25 to tyrosine was necessary to suppress FADD aggregation in solution (see also Eberstadt et al. [1998]), the simplest explanation for the role some amino acids play between F25 and K35 was that they mediated FADD-FADD interaction in a functional context. To test this hypothesis, Flag-tagged full-length FADD was coexpressed with either HA-tagged full-length FADD or the corresponding individual FADD domains in HEK293 cells. CoIP with FADD could only be achieved with full-length FADD or the FADD DED in this context (Figure 5A). Thus, the FADD DED mediates self-association. To identify the surface of self-association, mutants were introduced into full-length FADD, and CoIP was performed between wild-type, Flag-tagged FADD, and mutant HA-tagged FADD (Figure 5B). This experiment revealed that F25 and K33 form part of a FADD selfassociation surface. Other regions, in particular those in the RXDLF motif (Hill et al., 2002) ina6 (R71, D74, R77) and along the procaspase binding surface (D44), do not impinge upon FADD-FADD interaction in this assay. One of these tested positions, R71, has been implicated in modulating the interaction between receptor and FADD (Thomas et al., 2002, 2004; Supplemental Data), and several mutations in the RXDLF motif between residues 72 and 76 (R72A, R72A/D74A, or L75A/ L76A) disrupt the FRET response between FADD molecules overexpressed as YFP or CFP fusions in cell culture (Muppidi et al., 2006). The disruption in FRET led to the suggestion that it was the RXDLF motif that was the site of self-association in FADD (Yang et al., 2005; Muppidi et al., 2006), in contrast to our results (Figure 5B). To resolve this discrepancy, the impact of RXDLF mutation was examined by NMR spectroscopy in either the FADD DED or the FADD NMR construct (residues 2 191). The NMR spectrum of F25Y/K33E DED (Figure 5C) illustrates the following: (1) the pattern of peaks is similar for F25Y and F25Y/K33E, indicating a similar threedimensional fold for the two species; (2) chemical shift changes in F25Y/K33E are localized to the site of the mutation, suggesting only a local impact of the mutation on the protein structure; and (3) minimal changes in line width are observed for residues in other regions of the domain, indicating a minimal effect of K33E on the global fold of the domain. By contrast, the F25Y/R71E DED mutant displays a significant number of exchange-broadened peaks in the NMR spectrum and substantial chemical shift changes throughout the domain (Figure 5C). Although the similarity in peak pattern of F25Y/R71E relative to F25Y DED suggests that the F25Y/R71E domain is folded, the broadened lines indicate that the mutation induces dynamic disorder in the DED. The R72A mutation, which was sufficient to disrupt the FRET response between FADD-CFP and FADD-YFP in cell culture (Muppidi et al., 2006), had a more global impact on the protein structure. Introduction of F25Y/R72A into the NMR construct (residues 2 191) demonstrates that FADD is substantially disordered. Many NMR peaks exchange broaden, and none of the DED resonances are visible (Figure 5D), even though the protein was physically intact by SDS-PAGE (Figure 5D, inset) and retained much of its helicity as measured by CD (Figure 5D). Since several and cyan the least shifted signals. Note that the sample of F25Y displays some unfolded impurity as weak, sharp signals at w8 ppm that are not seen in samples of the mutant. (D) Overlay of NMR spectra for FADD (residues 2 191) F25Y versus F25Y/R72A. Exchange-broadened signals of F25Y/R72A in the DED are indicated by circled residues/labels for peaks in the F25Y spectrum. SDS-PAGE indicated that F25Y/R72A is intact (see inset). Shown in blue are selected residues in helical regions of the DD, which appear in the same positions in both spectra, suggesting that at least parts of the protein are folded. CD spectra of the FADD F25Y and F25Y/R72A (residues 2 191) NMR samples are shown to the right, indicating a similar helical content for the two proteins.

8 Molecular Cell 606 Figure 6. Stabilization of the DED Fold Stereo view of the 1.2 Å X-ray crystal structure of DED2 from MC159 (Yang et al., 2005). Residues forming salt bridges stabilizing the a1/a2 and a5/a6 helix-loop-helix elements are shown as sticks and labeled. The equivalent residues in the FADD DED are labeled with parentheses. Evidence for a hydrogen-bonded Arg side chain is observed in NMR spectra of the DED of FADD and PEA-15, wherein a single Arg N 3 proton is observed at 9 ppm, indicative of hydrogen bonding to an Arg side chain (data not shown). A trio of hydrophobic residues contributes to this charge-stabilized interaction. All six of these residues are highly conserved in the sequences of the FADD, procaspase, PEA-15, and MC159 DEDs (B). Blue shading indicates the six residues displayed in (A), green shading the additional residues forming part of the RXDLF motif and residues in a2 contributing to the stabilization of the domain fold. For comparison purposes, the hydrophobic patch is shaded yellow. peaks for helical residues in the FADD DD are unchanged in chemical shift (Figure 5D), at least some of the F25Y/ R72A protein retains its original tertiary structure. The loss in FADD self-association (Muppidi et al., 2006) or function (Yang et al., 2005) by mutation within RXDLF, therefore, most likely occurs secondary to a loss in structural integrity. The R71E and D74A mutations have a lower impact on overall protein order, accounting for the observation that self-association is still observed between wild-type and either the R71E or D74A mutant proteins in the CoIP experiment (Figure 5B). The role RXDLF plays in stabilizing the protein fold can readily be appreciated from the 1.2 Å crystal structure of v-flip MC159 (Figure 6)(Yang et al., 2005). Discussion A Diverse Organization for Tandem Death Motifs The three-dimensional structure of intact FADD reveals a tandem DED and DD oriented in an orthogonal, tailto-tail fashion. FADD exists in an extended conformation, leaving its surfaces for interaction with CD95, with the procaspases, and with itself sterically unhindered (Figure 3). The extended structure of FADD is contrasted by the more compact structure of tandem DEDs observed in the v-flip MC159 (Figure 7A) (Li et al., 2005; Yang et al., 2005). A predominantly hydrophobic interface is observed between DED1 and DED2 of MC159, which includes burial of F30 (equivalent to FADD F25) at the DED interface. Given the sequence similarity between the FADD and MC159 DEDs (>50%), one wonders why FADD isn t structurally more similar to MC159. The principal difference between the two proteins appears in a6 in FADD and in the corresponding a6/a7 helices in MC159 DED1. MC159 a6 (M71-F77) and a7 (K81- E86) are orthogonal and fold back on one another to form a series of extensive contacts with a5 in the domain. This pulls the irregularly structured portion of the linker (L87 L93) up toward DED1, inserting F92 into a pocket formed by three helices of DED 1 (a2, a4, and a5) (Figure 7A). FADD both lacks the hydrophobic residues found in the interdomain linker of MC159 and displays a continuous a6 helix from K73 to A86 (Figure 7A). Thus, FADD would be unable to form a DEDto-DD arrangement homologous to the tandem DEDs in MC159. A Model for the FADD/Procaspase-8 Complex The sequence similarity between the DEDs of MC159 and FADD suggests that the structure of MC159 might provide an insight into how FADD engages one or more DEDs in the procaspase prodomain. Residues from helices a2 and a5 in DED1 engage residues in helices a1 and a4 of DED2 in MC159 (Figures 7A and 7B) (Li et al., 2005; Yang et al., 2005). The procaspase binding-deficient mutants in the FADD DED reside in a1 and a4 and overlap with residues of MC159 DED2 forming the interface with DED1 (Figure 7B), suggesting that the FADD DED may be homologous to MC159 DED2 in its approach to a procaspase prodomain. This implies that one of the DEDs of the procaspase prodomain should be equivalent to MC159 DED1. To meet this criteria, a prodomain DED must display two hydrophobic residues at the interdomain interface in MC159, namely F30 and L31. The DEDs of procaspase-8 display a pair of conserved hydrophobic residues at structurally

9 FADD Interaction with Procaspase Figure 7. A Model for the Interaction between FADD and Procaspase-8 (A) The structure of FADD is compared with v-flip MC159, illustrating the structural distinctions between these proteins (see text). The insertion of the linker residue F92 in MC159 into DED1 is indicated in red and can be compared to the position of P92 in FADD (also red). (B) The structure of MC159 is shown with DED2 in cyan and DED1 in green. Highlighted in DED2 are helices a1 (red) and a4 (orange). Yellow bands represent positions in MC159 DED2 that are structurally equivalent to S12, R38, D44, and E51 in FADD. Blue bands highlight residues in MC159 DED2 that form contacts with DED1 (E105, R135, and N145) (Li et al., 2005; Yang et al., 2005). These comparisons suggest that the procaspase-8 binding surface of the FADD DED, formed by the a1 and a4 helices, engages procaspase-8 in a manner that is related to the interaction between MC159 DED2 and DED1. The FADD DED would be equivalent to MC159 DED2 (see text). (C) A docking model for FADD bound to procaspase-8, constructed with the program HADDOCK (Dominguez et al., 2000; seefigure S4). The resultant model has been colorized to highlight the a1 and a4 helices as in (B). Arrows emphasize the helix orientation at the FADD/procaspase-8 and procaspase-8 DED1/DED2 interfaces. Note that the a1/a4 helices in FADD are nearly perpendicular to the a1/a4 helices in procaspase-8 DED2. While the model suggests that the orientation of the FADD DED and DD remains unchanged in the presence of procaspase-8, this has yet to be determined. equivalent positions to MC159 F30 and L31: F24/L25 in DED1 and F122/L123 in DED2. However, only the residues in procaspase DED2 would be accessible for an interaction with FADD since F24 and L25 in procaspase DED1 would be buried at the DED1/DED2 interface. We conclude, therefore, that the procaspase-specific binding surface in FADD engages procaspase-8 DED2 in the vicinity of F122 and L123 (Figure 7C). Mutagenesis at these positions of procaspase-8 disrupts CoIP between FADD and procaspase-8 from 293T cells (Yang et al., 2005), in support of our view. The model constructed on the basis of the structure/function analysis (see Figure S4) suggests that the procaspase-8 prodomain would be oriented perpendicular to the FADD DED (see arrows in Figure 7C). While the model implies that the long axis of FADD and the prodomain are perpendicular to one another, we do not know if any reorientation of the FADD domains occurs upon binding CD95 or procaspase-8. An Emerging Picture of the DISC The structure/function correlates described for FADD and the structure of the v-flip MC159 (Li et al., 2005; Yang et al., 2005) lead to a convergent hypothesis for the interaction between FADD and procaspase-8. FADD DED mutants specifically deficient in the interaction with procaspase-8 structurally superimpose with residues at the interface between the two DEDs in MC159. This observation argues strongly that FADD should engage a procaspase-8 DED in a manner that is topologically similar to the DED-to-DED interaction in MC159. A similar hypothesis led to the identification of mutants in procaspase-8 DED2 that interfere with FADD binding (Yang et al., 2005). It is now apparent that the FADD DED is required to be covalently attached to the DD for a stable interaction with CD95 (Sandu et al., 2006). The observation that an exposed hydrophobic patch (Eberstadt et al., 1998) in the vicinity of F25 mediates FADD self-association

10 Molecular Cell 608 (Figure 5B) resolves a long-standing conundrum on the role of this patch in FADD function. Mutations within this patch disrupt all FADD functions without simultaneously disrupting the protein fold (Figure 5). By contrast, mutations in a5 and a6, which include the RXDLF motif (Hill et al., 2002; Kaufmann et al., 2002), lead to a loss in structural integrity for FADD (Figures 5C and 5D) as well as for the DED of the phosphoprotein enriched in astrocytes, PEA-15 (Hill et al., 2002). Although FRET suggested that FADD self-association is mediated by the RXDLF motif (Muppidi et al., 2006), NMR spectroscopy demonstrates that mutation of this motif is not tolerated by either PEA-15 (Hill et al., 2002) or FADD (Figure 5). Loss of function for the RXDLF mutants appears to be the consequence, therefore, of dynamic disorder introduced into the FADD DED. It is interesting to note that, while FADD and PEA-15 both contain the RXDLF motif and share 55% sequence similarity between their DEDs (Figure 6B), PEA-15 does not self-associate (Hill et al., 2002). FADD, on the other hand, self-associates via the hydrophobic patch in the vicinity of F25 (Figure 5), a feature absent in the DED of PEA-15 (Figure 6B). The emerging picture of the DISC as a complex of simultaneous FADD-FADD and FADD-receptor interactions can now explain why FADD does not spontaneously activate cell death in the absence of a ligand stimulus. Although FADD, procaspase-8, and procaspase-10 are constitutively present in cells, the affinity of FADD/CD95 or FADD/FADD interactions must be relatively low in isolation. This explains why the interaction between isolated FADD and CD95 DDs is neither easily reconstituted (Berglund et al., 2000) nor seen by CoIP from cells expressing the FADD DD (Zhang and Winoto, 1996; Sandu et al., 2006). To assemble a DISC requires a membrane-localized, activated receptor, in which the receptors cluster into higher order states in a FADDdependent manner (Siegel et al., 2004; C. Wu, C.S., and M.H.W., unpublished data). Since the procaspases appear to be recruited to the DISC as a preformed dimer (Boatright et al., 2003), the building block of the DISC should require two molecules of FADD, one for each molecule of procaspase. Thus, we speculate that a FADD homodimer forms at, or between, activated DRs to create the structural context necessary for procaspase recruitment and activation. This may explain why FADD/FADD and FADD/CD95 interactions are simultaneously required for productive DISC assembly. Given the close sequence similarity between c-flip, procaspase-8, and procaspase-10, it is likely that they will each bind the same surface of the FADD DED identified in this study. MC159 appears to be an exception, for it does not appear to compete with procaspase-8 for FADD binding sites in a pull-down assay (Yang et al., 2005), and MC159 must simultaneously associate with TRAF3 to act as an inhibitor of apoptosis (Thurau et al., 2006). Experimental Procedures Proteins and Nucleic Acids The design, construction, and expression of FADD and FADD mutants for protein interaction assays, NMR spectroscopy, and cellbased assays were conducted as previously described (Hill et al., 2004; Sandu et al., 2005). All of the FADD mutants tested for either self-association (Figure 5) or interaction with procaspase-8 (Figure 4) were previously studied for their effect on CD95 (Hill et al., 2004) or TRADD interaction (Sandu et al., 2005). NMR samples contained mm protein at ph 6.8 in either 10 mm sodium phosphate/ 300 mm NaCl or in 200 mm sodium phosphate buffer. Both buffers contained 1 mm benzamidine HCl, 1 mm DTT, and 50 mm NaN 3. For the mutations in the FADD DED (residues 2 88), the protein was expressed from pet28b and purified by Ni 2+ -chelate chromatography. NMR samples were prepared in 50 mm potassium phosphate (ph 4), 150 mm NaCl, and 1 mm DTT as previously described (Eberstadt et al., 1998). The F25Y/R72A mutant DED was poorly expressed at 20ºC or 37ºC, unstable at neutral ph, and found to be unfolded at ph 4 in the NMR tube; hence, this mutant was expressed as residues from pqe9 (Qiagen) with an N-terminal His 6 tag in the F25Y background and assessed under the same conditions used for the structure determination. Cell Culture and Transient and Stable Transfection Transient transfection of MCF7-Fas cells (Jaattela et al., 1995) with FADD and FADD D44R was performed as previously described (Hill et al., 2004). Jurkat I2.1 cells were stably transfected with various FADD constructs as previously described (Thomas et al., 2002, 2004; Sandu et al., 2005). Briefly, cells were transfected with 40 mg of PvuI linearized pcdna3.1-puro(+) vector encoding different FADD mutants/constructs. The cells were electroporated using a Bio-Rad Gene Pulser at 270V and 960 mf, and the cells were selected for puromycin resistance. The expression of FADD constructs was tested by Western blotting with rabbit anti-fadd polyclonal antibody (Santa Cruz Biotechnology) or an anti-ha peroxidase (3F10, Roche Diagnostics GmbH). Jurkat I2.1 cells were transiently transfectedwithdmrie-creagent (Invitrogen) using 4 mg ofanexpression vector individually encoding GFP and FADD or FADD mutant. Apoptosis Assay For stably transfected cells, Jurkat cells/ml were treated with an agonistic antibody (CH-11, 150 ng/ml) for 3 hr at 37ºC. Cells were stained with the FITC-conjugated Annexin V (FITC-Annexin V) and propidium iodide (Sigma-Aldrich). Cells ( ) were acquired and analyzed on a FACSCalibur flow cytometer (Becton Dickinson) using the CellQuest software (BD Biosciences). Apoptotic cells were quantified as the percentage of cells stained with Annexin V. For transiently transfected cells, the cells were stained with AnnexinV-Cy3, and GFP-positive cells were counted 18 hr posttransfection and 3 hr after activation with CH-11 (150 ng/ml). Coimmunoprecipitation IP of CD95 with FADD from MCF7-Fas cells was performed as previously described for overexpressed cells (Hill et al., 2004). IP of CD95 with FADD mutants from Jurkat I2.1 cells was performed by suspending cells stably expressing FADD or FADD mutants in 3 ml of RMPI medium (2FBS) and stimulated with 3 mg of CH-11 anti-human CD95 IgM for 10 min at 37ºC. The cells were harvested and washed with cold PBS and resuspended in 1 ml of lysis buffer, incubated on ice for 10 min, and cleared by centrifugation. Nine hundred microliters of supernatant was mixed with 40 ml of goat anti- IgM-agarose beads (Sigma-Aldrich) and nutated for 3 hr at 4ºC. The beads were washed five times with lysis buffer and resuspended in 30 ml of SDS sample loading buffer prior to SDS-PAGE. Circular Dichroism CD spectra were acquired with an AVIV model 62DS spectrometer, using a 0.1 cm path-length cell. The NMR samples of Figure 5D were diluted with 50 mm sodium phosphate (ph 7.0) to 8 mm final concentration. Spectra were acquired between 195 and 260 nm in 1.0 nm steps with a 3 s delay time for three scans at 15ºC. The data were baseline corrected by solvent subtraction. Molar ellipticity was calculated from the average of reported ellipticity of the three scans using the equation ([Q] = 3298 D3). NMR Spectroscopy All NMR experiments were acquired at 15ºC using either a Bruker DMX 600, Avance 600, or Avance 800, all equipped with a cryoprobe. RDCs were measured in the NMR sample buffer to which the

11 FADD Interaction with Procaspase filamentous phage Pf1 was added to a final concentration of w9 mg/ ml (Hansen et al., 1998) or by 1:1 dilution of a 10% stock solution of DLPC/CHAPSO/CTAB (4.2:1:0.1 molar ratios) (Wang et al., 1998) with FADD stock solution (1 mm). Initial values for the axial component of the dipolar coupling tensor and for the rhombicity were determined from a normalized histogram of the RDCs (Clore et al., 1998). NMR spectra were processed with NMRPipe/NMRDraw (Delaglio etal., 1995) andanalyzedusing PIPP andstapp (Garrett et al., 1991). Structure Calculations The structures were calculated with the program X-PLOR-NIH adapted to incorporate pseudopotentials for 3 J NHa coupling constants (Garrett et al., 1994), secondary 13 C a and 13 C b chemical shifts (Kuszewski et al., 1995), a conformational database potential for dihedral angles (Kuszewski and Clore, 2000), and a harmonic potential for the refinement against RDCs (Tjandra et al., 1997). Initial refinement began from an extended polypeptide chain and the fold established with the unambiguous set of NOEs, F and J angles extracted from TALOS (Cornilescu et al., 1999), and 3 J NHa couplings constants. The two interdomain NOEs and/or the RDCs measured in Pf1 bacteriophage were included in separate folding trials as described in Supplemental Data. These folding experiments established that the interdomain NOEs and RDCs reinforced one another rather than biased the initial model, leading to a single model following iterative rounds of NOE analysis and refinement employing a torsion angle restraint protocol provided in the Xplor-NIH distribution. Both sets of RDCs were included following the initial folding trials with the force constant set to a final value of 1.0. The agreement between expected and calculated RDC values was determined by SVD analysis with the program DC (Delaglio etal., 1995). Structure quality was assessed with PROCHECK and PROCHECK_NMR (Laskowski et al., 1996). Structures were displayed and analyzed using the program PYMOL (DeLano 2002). Supplemental Data Supplemental Data include supplemental text and four figures and can be found with this article online at content/full/22/5/599/dc1/. Acknowledgments We thank Vishva Dixit, Michael Lenardo, and Marcus Peter for cdnas of human CD95, FADD, and caspase-8; and Marcus Peter for MCF7-Fas cells and antibodies. We further would like to acknowledge Yigong Shi, Hao Wu, David Fushman, Ranajeet Ghose, Joel Tolman, and G. Marius Clore for many useful discussions or the sharing of unpublished results over the course of this work. This work was supported by fellowships from the Human Frontier Science Program, the NHMRC of Australia, and the Norman and Rosita Winston Foundation (to J.M.H.) and by the National Science Foundation, the National Institutes of Health, and Irma T. Hirschl Trust grants (to M.H.W.). 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